1. Field of the Invention
This invention relates to a method to analyze in vivo oxidative stress status in an organism by measuring glucuronidated forms of isoprostanes isolated from the organism and determining a total isoprostane profile including the glucuronidated form.
2. Description of Related Art
Oxidative stress, i.e. free radicals derived from oxygen, when occurring in vivo in biological systems, including cells and organisms, disrupts cellular structures and functions and contributes to disease development and aging. It would be useful to determine the oxidative stress level in vivo so as to provide a measure of the potential for disease development Halliwell, et al 1987!.
Isoprostanes are a novel class of prostaglandin-like compounds formed in vivo or in vitro by a non-cyclooxygenase-dependent free-radical catalyzed mechanism Morrow, et al, 1990a!. In normal humans, levels of free (non-conjugated) form of 8-epi-prostaglandin F.sub.2.alpha., (8EPGF2) measured by gas chromatograph and mass spectroscopy (GC/MS) provide a normal range from 5-50 pg/mL plasma and 500-3,000 pg/mg urinary creatinine, respectively Morrow, et al, 1990a; 1990b; Morrow and Roberts, 1991!. The in vivo concentration of 8EPGF2 increases dramatically in animal models of lipid peroxidation Awad, et al 1994!, a model oxidative stress.
F.sub.2 -isoprostanes are formed from bicycloendoperoxide intermediates. Analogous to cyclooxygenase derived compounds the endoperoxide intermediate products of prostaglandin synthase (prostaglandin G.sub.2 and prostaglandin H.sub.2) are unstable and can also undergo non-enzymatic rearrangement to PGD.sub.2 and PGE.sub.2 (t1/2=.about.5 minutes in aqueous solution). Therefore, the possibility that the bicyclic endoperoxide precursors of 8EPGF2 undergo rearrangement to D-ring and E-ring isoprostanes was investigated and found that the D.sub.2 /E.sub.2 -isoprostanes are in fact formed in vivo in addition to F.sub.2 -isoprostanes Morrow, et al 1994!. In contrast to 8EPGF2, free D.sub.2 /E.sub.2 -isoprostanes were not detectable (&lt;5 pg/mL) in the circulatory system of untreated rats, but were found at significant levels in rats treated with CCl.sub.4 to generate free radical induced lipid peroxidation Morrow, et al 1994!.
Therefore, measurement of isoprostane concentrations correlate with free radical production and this measurement has significant diagnostic potential for assessment of oxidative stress and disorders such as hepatorenal syndrome Awad, et al, 1994!, atherosclerosis Morrow, et al, 1995!, and carcinogenesis Takahashi, et al, 1992!. High concentration of serum 8EPGF2 was found, in part, to be responsible for the kidney failure due to the intense vasoconstriction of renal arteries in hepatorenal syndrome patients Awad, et al, 1994!. Free isoprostanes have been measured in plasma and urine as a relative measure of oxidative stress in vivo.
However, in any assay for isoprostanes all forms of the isoprostanes need to be considered and their metabolites may need to be considered. Assays for the free form are known. However, there is a concern about measurements of only the free (non-conjugated) form being completely representative. A significant portion Morrow et al, 1997! of the free form in urine is probably derived locally from the kidney and does not represent systemic oxidant stress. For other body fluids isoprostanes may also be esterified to phospholipid Morrow et al, 1997!, and both free and esterified increase in oxidant stress. Therefore it is apparent that measurement of only the free form may provide an incomplete picture of oxidant stress in vivo.
8EPGF2 has a vasoconstrictor property Morrow and Roberts, 1991; Morrow, et al, 1994!, which may be, in part, mediated by binding to the thromboxane receptor Fukunaga, 1993! or a distinct 8EPGF2 receptor Rich and Singh, 1979!. Thus, any structural change of 8EPGF2 due to esterification or conjugation will be expected to affect binding of the 8EPGF2 to the receptors.
Free isoprostanes have been measured by mass spectrometry Morrow, et al, 1990a; 1990b; Morrow and Roberts, 1991; Awad, et al, 1994!. This method is not well suited for routine clinical determinations due to:
(a) sample preparation for GC/MS is long and tedious, PA1 (b) this method assesses only free isoprostanes, and PA1 (c) it is not practical to measure all the possible
8EPGF2 metabolites using GC/MS. Recently, free 8EPGF2 in serum and urine specimens were measured using an ELISA. However this assay again does not provide a complete picture of oxidative stress since the assay does not measure all forms and metabolites of the isoprostanes which is necessary to provide a complete profile or better representation of oxidative stress in vivo.